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E Florvaag

Publications and source records attributed to E Florvaag.

14 recordsLinked to original sources
PubMed1991

The structural requirements of epitopes with IgE binding capacity demonstrated by three major allergens from fish, egg and tree pollen.

Three major allergens from cod fish, egg white and tree pollen, were characterized by studies on their allergenic and antigenic structures. The major allergen of cod fish, Allergen M "parvalbumins pI 4.75", is composed of 113 amino acid residues with a molecular weight of 12,328 daltons. It comprised three domains, AB, CD and EF, consisting of 3 helices interspaced by one loop. Each of the loops of the CD and EF domains each coordinates one Ca2+. The antigenicity and allergenicity of Allergen M was deduced from studying the modified protein and some particular synthetic peptides. Three sites were encompassing IgE binding epitopes namely peptides 33-44, 65-74 and 88-96. A novel peptide (49-64), of the CD-domain, was demonstrated to be allergenically/antigenically active and cross reactive with birch pollen allergen, which incidentally was used as a negative control. This site encompassed two repetitive sequences (D-E-D-K) and (D-E-L-K), suggested to be mutually critical for the specificity of antibody binding. This hypothesis was reconfirmed by SPPS of several analogous peptides of region 39-64. Furthermore, peptide 88-103 of the EF-domain was similarly synthesized; it functioned as a monovalent hapten, blocking and not eliciting allergic reaction. Moreover, peptide 13-32 of domain AB, the non-calcium binding domain, was thoroughly tested. The results of PK inhibition showed clear activity and the peptide was found to function at the level of a divalent determinant. Ovalbumin (OA) is the most dominant of five major allergens of egg white and universally used as model protein. OA allergenic epitopes were shown to be mainly determined by the primary structure and depend on certain peptide chain length. The N-terminal decapeptide (OA 1-10) was shown to react with reaginic IgE. Direct skin test on egg allergic patients, showed no activity and the site was therefore concluded to encompasses one single Ig binding haptenic epitope. Peptide OA 323-339, was demonstrated to be valuable in studies of T-cell recognition of protein antigens. Three analogous peptides of this region were prepared and clearly shown to be immunogenic in rabbits and to bind specific IgE from patients allergic to egg. OA 323-339 was concluded to encompass an allergenic and antigenic epitope which was recognized by human and rabbit B-lymphocytes. Eight peptides in the region 11-122 were similarly synthesized. A test battery was performed to study this region using rabbit polyclonal antibodies and human specific IgE. Some of these sites were involved in binding of particular Ig paratopes. Five immunogenic peptides from the major allergens of tree pollen extracts (segment 23-38), were synthesized. The selection of those peptides was setteled using two algorithms for providing the optimal hydrophobicity.(ABSTRACT TRUNCATED AT 400 WORDS)

Allergens↗
PubMed1988

A comparison of the antigenic and allergenic components of birch and alder pollens in Scandinavia and Australia.

Allergens in birch (Betula) pollens from B. pendula grown in Australia and Norway, B. davurica and B. populofolia and from alder (Alnus incana) were identified by electroblotting, following separation by SDS-PAGE, transfer to nitrocellulose membranes and incubation with sera from birch pollen-allergic subjects. Of 42 antigenic components detected by protein staining in the pollen extract from B. pendula grown in Norway, 17 bound IgE. The allergenic components included those already reported in the literature at MWs of 40, 29, 25, 17 and 10-12 kd, as well as previously undescribed components at MWs of 90, 79, 60, 50, 38, 35, 31, 27, 23, 16, 15 and 14 kd. The major IgE-binding components were located in the low MW region 10-17 kd for all species of birch and alder pollen proteins studied. Results reported here provide the first evidence of birch and alder pollen allergies in Australia. Extensive heterogeneity was observed amongst the sera from birch pollen-allergic subjects in both Norway and Australia. Cross-reactivity appears to exist among the proteins present in pollen from the various birch species and from alder.

Australia↗
PubMed1988

Comparative studies on tree pollen allergens. XIV. Characterization of the birch (Betula verrucosa) and hazel (Corylus avellana) pollen extracts by horizontal 2-D SDS-PAGE combined with electrophoretic transfer and IgE immunoautoradiography.

The crude aqueous extracts of birch (Betula verrucosa) and hazel (Corylus avellana) pollen were characterized by horizontal isoelectric focusing (IEF) and one-dimensional and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1-D and 2-D SDS/PAGE). Isoelectric focusing reference patterns obtained in pH 3.5 to 9.5 gradient gels contained 31 and 41 bands while the 2-D patterns consisted of 51 and 72 protein spots, respectively. Electrophoretic transfer to nitrocellulose membranes and subsequent 125I-immunoautoradiography demonstrated IgE binding in the region between pH 4.8 and 5.4 for both extracts. The main allergenic reactivities of this pH region were distributed by SDS/PAGE to the molecular weights of 29.5, 17, and 12.5 kilodaltons for birch and 15.5 and 12 kilodaltons for hazel. The high resolving power and the independence of rabbit antibodies were considered the major advantages of the described 2-D methodology as compared with conventional crossed immunoelectrophoretic and radioimmunoelectrophoretic techniques.

Animals↗
PubMed1987

Comparative studies on tree pollen allergens. XII. Evaluation of direct and indirect autoradiography systems for 125I in crossed radioimmunoelectrophoresis and other immunoautoradiographic techniques.

Visualization by autoradiography of specific IgE binding in crossed radioimmunoelectrophoresis (CRIE) and other 125I-immunoautoradiography (IAR) techniques is done in two different ways; either by traditional direct autoradiography (D-ARG), where the film is exposed to the 125I-anti-IgE incubated sample at room temperature, or by indirect autoradiography (ID-ARG), applying intensifying screen, low-temperature exposure and, eventually, pre-exposure. This study confirmed that D-ARG provided the benefits of simplicity and better image resolution with the disadvantage of prolonged exposure periods. ID-ARG reduced the exposures needed to produce film image densities of 0.01 and 0.1 A540 nm (i.e. autoradiographic sensitivity (AR sigma) and autoradiographic speed (ARs] to 1/18 and 1/55 respectively of the corresponding exposures in D-ARG. The lowest detection limits for 125I in 24 h were 1.2 cpm mm-2 with the indirect and 6.8 cpm mm-2 with the direct systems investigated. The major drawbacks of ID-ARG were inferior image resolution and higher background levels, especially when pre-exposure was included.

Allergens↗
PubMed1987

Comparative studies on tree pollen allergens. XVII. Immunochemical analysis of the international standardization extracts of birch (Betula verrucosa) pollen as compared with a local partially purified extract.

Six different birch pollen extracts were analyzed by 20 laboratories for the standardization of birch (Betula verrucosa) pollen extracts used for diagnosis and specific therapy of patients with birch pollen allergy. The extracts were collected and delivered by the International Union of Immunological Societies, Allergen Standardization Subcommittee. One of the extracts, designated M, was proposed as an international standard (IS)-candidate of birch pollen extracts. The protein content of the IS candidate M was found to be 1.12 mg/mL, more than 2-fold higher than any of the other extracts analyzed. This preparation was among the extracts containing the highest number of protein components, as shown by isoelectric focusing, 28 lines, and by 11 precipitates in crossed immunoelectrophoresis. The allergenic reactivities were tested by crossed radioimmunoelectrophoresis (CRIE) and by radioallergosorbent test (RAST)-inhibition. In CRIE, the proposed IS (M) showed similar affinity for binding patients' IgE as the other extracts, as judged by the autoradiographic illustrations. Except for extract L, the values of RAST-inhibition for the rest were very similar. An IS extract should qualify for the criteria suggested for an optimal allergen preparation, containing minimal amounts of non-allergenic antigens and providing quantitatively and qualitatively all the allergenic proteins. The appropriateness of this selection seems unjustified in view of the above criteria.

Allergens↗
PubMed1986

Antigenic and allergenic determinants of ovalbumin. I. Peptide mapping, cleavage at the methionyl peptide bonds and enzymic hydrolysis of native and carboxymethyl OA.

The effects of enzymic cleavage and perturbing the conformation of the allergenic and antigenic determinants of hens egg white albumin (OA) were examined. Hens egg white extract of a total protein concentration 8.43 g/l was prepared. Isoelectric focusing in sodium dodecyl sulfate and polyacrylamide gel peptide maps for the crude egg white extract showed 26 spots visualized by staining with Coomassie blue. The OA was purified using a TSK-2000 gel filtration chromatography column. The specific allergenic reactivity of the purified OA as measured by RAST inhibition and direct RAST was relatively high: 3 micrograms gave an inhibition of approximately 10%. The cleavage of OA with cyanogen bromide resulted in 4 fractions, all capable of binding specific IgE with the first peak showing the highest inhibition. Thermal denaturation of OA had no direct effect on the antigenic reactivity. RAST inhibition values for the denatured protein were similar to those of the native protein. Carboxymethylation of OA gave a product with only 20% of the inhibition reactivity. Further treatment with trypsin did not abolish the allergenic and antigenic reactivities as shown by RAST inhibition and by deflection of OA line in rocket line immunoelectrophoresis. On the other hand, limited pepsin hydrolysis destroyed the antigenic structure of the molecule. The reactivity of OA is thus relatively stable and could easily be retained making it possible to identify the allergenic determinants of enzymic hydrolysates used for elucidating the antigenic structure of the molecule.

Allergens↗
PubMed1986

Comparative studies on tree pollen allergens. XIII. Partial characterization of the alder (Alnus incana) pollen extract by two-dimensional IEF/SDS-PAG electrophoresis combined with electrophoretic transfer and immunoautoradiography.

Horizontal isoelectric focusing (IEF) and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-D SDS-PAGE) combined with electrophoretic transfer (ET) and immunoautoradiography (IAR) were used to characterize the crude alder (Alnus incana) pollen extract (AI crude) and fraction AI 34. IEF resolved AI crude and AI 34 into 34 and 39 distinct protein bands, respectively, when stained with Coomassie brilliant blue. The band patterns were similar and the majority of bands were located in the pH region 4.0-7.0. After ET and IAR, the distribution of antigenic reactivities was similar to that of the protein stain. Allergenic reactivity was detected in the pH region 4.6-5.2 only, where two bands at pH 4.9 demonstrated dominating IgE-binding properties. The 2-D reference maps of AI crude and AI 34 consisted of 40 and 45 protein spots, respectively. The autoradiogram of AI crude demonstrated 35 spots capable of binding rabbit IgG. The main IgE-binding zone was located at pH 4.6-5.2 and 20 kilodaltons with an area of weaker autoradiographic signals at pH 4.6-5.2 and 40 kilodaltons. ET from polyacrylamide gels to nitrocellulose (NC) membranes was quantitative and IAR gave significant radiostaining of spots containing 0.01 and 0.1 microgram of AI crude in the IgG and IgE systems, respectively. For the characterization of the alder pollen extract, horizontal 2-D SDS-PAGE combined with ET and IAR provided a powerful supplement to conventional crossed immuno- and radioimmunoelectrophoretic techniques (CIE/CRIE).

Animals↗
PubMed1984

Comparative studies on tree pollen allergens. VIII. Immunological properties of the alder (Alnus incana) pollen extract.

The immunological properties of the aqueous crude alder pollen extract (AI crude) and gel filtration fractions AI 3, AI 4 and AI 34 (pool of fractions AI 3 and AI 4) were examined by immuno- and radioimmuno-electrophoretic techniques, RAST titration, RAST inhibition and skin prick tests (SPT). In CIE, the AI crude extract and AI 34 displayed reference precipitate patterns consisting of 27 and 24 visible Coomassie brilliant blue stained lines, respectively. The CRIE allergogram performed by incubation with 18 individual reaginic sera detected three IgE-binding antigens characterized by different IgE-binding properties. Antigen No. 7 (Ag 7) was demonstrated to be the major IgE antibody-binding antigen of alder pollen, while Ag 1 and Ag 11 were classified as intermediate allergens. The allergens of alder pollen were located in fractions AI 3 and AI 4 of the gel filtration chromatogram. Ag 7 was present in both fractions as demonstrated by FRIE with autoradiography (FRIEWA) on the gel filtration fractions and tandem-CRIE of AI 3 and AI 4. The CRIE allergogram, RAST, RAST inhibition and SPT demonstrated fraction AI 34 to be allergenically representative of the AI crude extract both qualitatively and quantitatively. Thus, fraction AI 34 was considered an optimal purified allergen extract of alder pollen, a suitable material for further biochemical characterization and trials on purification of the allergenic reactive antigens.

Animals↗